Dynamics and self-organization of membrane-bound minimal actin and actomyosin cortices based on ezrin-PtdIns[4,5]P2 linkage
by Nils Liebe
Date of Examination:2022-04-11
Date of issue:2022-11-22
Advisor:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Sarah Köster
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Burkhard Geil
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Nadja Jun.-Simeth
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EnglishThe cellular cortex is a thin actin layer attached to the cytoplasmatic plasma membrane leaflet of mammalian cells. Through a complex interaction with hundreds of actin binding proteins (ABPs), controlling e.g. the cortex organization and dynamics, the cortical actin network is a key regulator of cell morphogenesis, shape and motility. A crucial property associated with these functions is the direct membrane attachment by linker-proteins such as ezrin/radixin/moesin (ERM). The high complexity of the cortical actin network impedes the characterization of individual component properties and functions in vivo. Thus, a biomimetic minimal actin cortex (MAC) composed of solid-supported lipid bilayers, the constitutively active ezrin mutant T567D, and pre-polymerized filamentous actin (F-actin) was utilized to model the actin cortex plasma membrane linkage in a minimal component system. The impact of in vivo mimicking conditions such as the plasma membrane lipid composition, branched actin filaments and a crowded media was investigated in terms of the MAC organization and membrane attachment. It was demonstrated that the amount of membrane-bound ezrin and F-actin not only increases with higher concentrations of the ezrin receptor lipid L-α-phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2), but also significantly in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), which resembles the composition of the cytoplasmatic plasma membrane leaflet. In addition, it was shown that a methyl cellulose crowded media, mimicking the cytoplasma density, increases the amount of membrane-bound F-actin. Growing amounts of membrane-bound F-actin could further been shown to dictate both single F-actin organization and MAC architecture, switching from single to bundled filaments and entangled to nematic ordered MACs. In comparison to this unbranched actin, it could be revealed that the amount of membrane-bound F-actin is reduced by a co-polymerization with the actin related protein 2/3 complex, although dense 3D networks could be formed in solution. Atomic force microscopy indentation experiments on freestanding pore-spanning membranes (f-PSMs) revealed a reduced lateral membrane tension and a viscoelastic force response of f-PSMs with biomimetic attached MACs. Analyzing the reorganization of membrane-bound minimal actomyosin cortices as a function of the ezrin-PtdIns[4,5]P2 linkage and membrane composition, revealed that a lipid composition of 1 - 2 mol% PtdIns[4,5]P2 and 17 mol% POPS, resembling the inner plasma membrane leaflet, promotes the MAC contractility. These findings highlight that lipids which do not directly interact with the actin cortex, such as POPS, requires consideration in the design of actomoysin studies.
Keywords: minimal actin cortex; actomyosin; artificial membranes; ezrin-PtdIns[4,5]P2 linkage